Dissecting the molecular mechanisms of cellular heterogeneity controlling infection-associated development in plant pathogenic fungi

Projektinformationen

CELL-ENEITY

ID Finanzhilfevereinbarung: 101076478

DOI

10.3030/101076478

EK-Unterschriftsdatum 19 Februar 2023

Startdatum 1 Dezember 2023

Enddatum 30 November 2028

Finanziert unter

• European Research Council (ERC)

Gesamtkosten

€ 1 499 439,00

EU-Beitrag

€ 1 499 439,00

Koordiniert durch

UNIVERSIDAD PUBLICA DE NAVARRA
Spain

Periodic Reporting for period 1 - CELL-ENEITY (Dissecting the molecular mechanisms of cellular heterogeneity controlling infection-associated development in plant pathogenic fungi)

Berichtszeitraum: 2023-12-01 bis 2026-05-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Clonal microorganisms display cellular heterogeneity at the transcriptional level, to survive under unfavourable conditions or differentiate into specialised structures. This is the basis of antibiotic and fungicide resistance, but very little is known about how cellular heterogeneity originates and operates in the infection biology of agricultural fungi.
Magnaporthe oryzae is one of the most devastating fungal pathogens in the world that destroys enough rice to feed 60M people every year. It produces ~50,000 new spores a day from a single lesion in the fields, but it remains unknown whether they are transcriptionally different. Spores contain three cells that display cellular heterogeneity between them during appressorium development, a specialised cell necessary for infection. Two of the cells undergo autophagy rapidly and the third undergoes a mitotic division leading to the formation of the appressorium. The mechanism by which cellular heterogeneity operates in spores has never been elucidated.
This proposal will identify, for the first time, the molecular mechanisms driving cellular heterogeneity and genes subjected to it. An unparalleled resolution of the infection-associated developmental program of individual spore cells will be obtained by scRNA-seq, which will identify a cohort of virulence factors critical for infection. I propose that the underlying mechanism of cellular heterogeneity is the cell cycle, through the activity of Cyclin Dependent Kinases (CDKs) and a novel group called non-PSTARE CDKs, reported to be regulators of transcription in other organisms. By a state-of-the-art chemical genetic approach combined with phosphoproteomics, their role and signalling pathways will be e determined. Overall, with this proposal, novel components associated to the infection process of one of the most threatening fungal pathogens in the world will be determined, opening avenues that up to date have not been explored and whose potential is inestimable

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

Objective 1:
- Successful isolation of nuclei from mycelium, spores, in vitro appressorium structures, and in planta appressorium structures
- Successful nuclei quantification using flow cytometry and stablishment of nuclei integrity

Objective 2:
- Obtention of conditional and null mutants for the CDKs, CRISPR GFP fusions, and ectopic GFP integrations
- Cloning of CDKs to carry Y2H analysis to identify interactors
- Execution of IP-MS of CDKs

Objective 3:
- Successful stablishment of CRISPR to carry gene replacement, epitope tagging of genes of interest

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

For the first time, a succesful nuclei isotlation from spores and appressoria has been carried and we have demonstrated that those nuclei are valid and with the correct integrity. We have been able to identify cell clusters during appressorium development and annotate some of those. We have demonstrated that one of the PSTAIRE CDK called Cdc28, controls cellular heterogeneity within the spore. Spores of a mutant that is constitutively active for Cdc28, display cell identity re-configurations. The loss of the specific wild type configuration affects pathogenicity indicating that cellular heterogeneity determines the pathogenicity ability of this fungus. Moreover we have demonstrated that spore populations are transcriptionally heterogenerous, and we have identify a list of putative drivers, inlcuding those related with cell cycle, some transcription factors and chromatin remodellers.